Suppression of Dysplasia and Hyperplasia by Calcium in Organ-Cultured Urinary Bladder Epithelium

[CANCER RESEARCH 38, 586-592, March 1978]

Suppression of Dysplasia and Hyperplasia by Calcium in Organ-cultured Urinary Bladder Epithelium

David H. Reese and Rosalind D. Friedman

Lung Cancer Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014

ABSTRACT bladder epithelia to F12 medium is especially interesting because the development of endophytic growth is a char The cause for the development of endophytic growth acteristic feature of preneoplastic bladder epithelia that (nodular downgrowth) in rat urinary bladder epithelia cul have been exposed to known bladder carcinogens (2, 14, tured in protein-free medium was traced to a deficiency 19). Endophytic growth has been stated to be the most of Ca in the culture medium. Endophytic growth, which important histological feature of carcinoma in situ in blad is a characteristic in vivo histolÃ³gica! feature of the pre- ders of rats that received the bladder-specific carcinogen neoplastic phase of experimental bladder cancer, was /v-butyl-W-(4-hydroxybutyl)nitrosamine in the drinking water induced in vitro by 0.3 mm Ca2" but was suppressed by (14). It is not known, however, whether endophytic growth 1.8 rriM Ca; . Magnesium at 1.8 HIM had no effect on should be considered a precursor lesion to cancer, or endophytic growth. whether it should be considered merely a manifestation of Ca deficiency was also found to be a significant cause an unstable epithelium that may or may not eventually have of hyperplasia. Ca concentrations above 0.9 mmresulted a direct relationship to the development of cancer (see in a substantial inhibition of hyperplasia in the bladder Refs. 5, 13, and 19). From an experimental point, we think epithelium; a greater than 50% inhibition was observed with 1.8 rtiMCa2*. Conditions were found, however, which that the important aspect of endophytic growth, whether it is induced by carcinogen or is induced in vitro by F12 caused the suppression of endophytic growth without an medium, is that it represents a breakdown in the normal apparent suppression of hyperplasia. When the Ca2" concentration was decreased to 0.075 architecture of the epithelium in which cells have extended beyond the territories that they normally occupy. Since HIM and below, epithelial cells lost cohesiveness and invasive cancer is an extreme example of cells that have infiltrated into the stroma. Epithelial cells were observed extended beyond their normal territorial boundaries, it individually and in clusters within the lamina propria. would be of considerable significance to know why endo Evidence of lytic activity was also observed in the lamina phytic growth arises in the bladder epithelium and how this propria. Epithelial cells that infiltrated into the stroma particular pattern of growth can be suppressed. displayed occasional mitotic figures and frequently con In this paper we present an analysis of endophytic growth tained abnormally appearing nucleoli. in F12 medium and identify the cause of this growth to be a The ability of low Ca concentration in the culture deficiency of Ca2* in the culture medium. We also demon medium to induce epithelia to mimic some of the growth strate that Ca2+ deficiency is a significant cause of the patterns that appear during the development of experi hyperplastic response in organ-cultured bladder epithe mental bladder cancer in vivo, suggests that there may lium. be an early change in some aspect of cellular Ca2* in epithelia and/or stroma that have been exposed to blad der carcinogens. MATERIALS AND METHODS

INTRODUCTION Animals. Male Fischer 344 rats were obtained from the Charles River Breeding Laboratories, Wilmington, Mass., In earlier studies (16, 17) we demonstrated that the and used at 7 weeks of age. urinary bladder epithelium of the rat can be cultured in Organ Culture Procedure. Animals were anesthetized various media without the addition of serum or other pro with CO-,. Urinary bladders were removed aseptically and tein supplements. In MEM-EBS1 and Waymouth's MB752/ cut into equal halves longitudinally. Expiants were cultured, 1 medium, organ-cultured epithelia proliferated but re with the epithelium positioned dorsally, on stainless steel tained their characteristic transitional architecture. In grids in 60-mm plastic culture dishes containing 2 ml of Ham's F12 medium, on the other hand, extensive prolifera medium. The medium contained 100 units of penicillin and tion and dysplasia were observed. The most prominent 100 M9 of streptomycin per ml. Cultures were maintained dysplastic feature of bladder epithelia cultured in F12 me at 36Â°in a gas phase of 4.5% COj in air. Medium was dium was the development of endophytic growth (nodular changed every 2 or 3 days. Unless specified differently, all downgrowth) into the underlying stroma. This response of culture media components were obtained from Grand Is land Biological Co., Grand Island, N. Y. Calcium chloride and magnesium chloride were reagent grade and obtained 1 The abbreviations used are: MEM-EBS. Eagle's minimal essential me dium with Earle's balanced salts solution; MEM, Eagle's minimal essential from Baker and Adamson and Mallinckrodt, respectively. medium; EBS, Earle's balanced salt solution. Within each experiment, 4 to 6 expiant cultures were Received July 25. 1977; accepted December 5, 1977. prepared for each specific medium formulation investi-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Suppression of Dysplasia and Hyperplasia by Ca2 gated. Each experiment was repeated at least 2 times and usually less clearly defined than those of epithelia cultured in 1.8 mM Ca2". in some cases more than 5 times. Another feature of epithelia cultured in 1.8 mM Ca24,

RESULTS which was revealed most clearly when the epithelium be came separated from the stroma as in Fig. 4/4, was the Ca Suppresses Endophytic Growth. Our initial ap presence of extremely flattened cells lining the epithelial- proach in attempting to identify the cause of endophytic stromal border. These cells were not evident in those growth in F12 medium was to investigate whether the endophytic regions of epithelia cultured in 0.3 mM Ca24, in elimination of some of the growth-associated components which the epithelial-stromal borders were not clearly de present in F12 medium, such as putrescine and linoleic fined (Fig. 4B). Some were observed, however, in endophy acid, would eliminate endophytic growth. It was found tic regions in which the epithelial-stromal borders were that, although more growth occurred in medium with pu more clearly defined (Fig. 3/4), but these cells were not as trescine and linoleic acid than in medium without these compressed. components, endophytic growth still occurred in their ab Experiments were carried out to determine whether, by sence. increasing the magnesium concentration to 1.8 mM in the Rather than test all suspected components individually presence of 0.3 mM Ca2', endophytic growth could be for their ability to produce endophytic growth, we decided blocked, or if the elevated magnesium concentration inter to test the major groups of components such as the salts fered with the suppression of endophytic growth by 1.8 mM and amino acids of F12 medium for their ability to induce Ca2'. Magnesium was ineffective in both instances. Since endophytic growth when substituted for the same compo calcium chloride and magnesium chloride were used in nents of MEM-EBS which do not normally cause endophytic these experiments, these results also indicate that Ca24, growth. The first component tested, the salts component, and not CI , suppressed endophytic growth. proved to be the cause of endophytic growth. When one- Ca2* Suppresses Hyperplasia. In addition to demonstrat half of a bladder was cultured in MEM-EBS, growth was ing that 1.8 mM Ca24 suppressed endophytic growth, a nonendophytic (Fig. 1/4). When the other half of the same comparison of expiants cultured in low and high Ca2' also bladder was cultured in MEM containing the salts compo suggested that high Ca24 (1.8 mM) suppressed hyperplasia. nent of F12 medium, endophytic growth occurred (Fig. To verify these observations quantitatively, the extent of 10). hyperplasia was measured, by a previously described One of the major differences between F12 salts and EBS method (17), in epithelia that were cultured for 12 days in is the presence of copper, iron, and zinc in F12 salts. MEM with F12 salts containing Ca2' concentrations ranging These were obtained from 2 sources (Grand Island Biologi from 0.3 HIM to 1.8 mM. In these experiments, endophytic cal Co. and Johnson, Matthey and Co., London, England) growth was not as pronounced as that encountered in 14- and were tested in MEM-EBS for their ability to induce day cultures and was thus more amenable to quantitation. endophytic growth. None was effective, either singly or in The suppression of hyperplasia by Ca2' was dose depend combination with others, in stimulating endophytic growth. ent (Chart 1); a substantial suppression was observed when Another major difference between F12 salts and EBS is the Ca2' concentration was increased above 0.9 mM. In in the Ca2+ concentration; F12 salts contain 0.3 mM Ca2' medium containing 1.8 mM Ca2', the extent of hyperplasia while EBS contains 1.8 mM. Experiments were carried out was less than 50% of that occurring in medium containing to determine whether, by elevating the Ca24 level in F12 0.3 mM Ca2'. salts, endophytic growth could be blocked. When one-half Since higher Ca21 levels suppressed hyperplasia in addi of a bladder was cultured in MEM with F12 salts, endo tion to endophytic growth, it could be implied that endophy phytic growth developed (Fig. 2A). Endophytic growth tic growth occurs as a direct result of excess hyperplasia. was suppressed, however, in the other half of the same Two lines of evidence demonstrate that this is not necessar bladder cultured in MEM with F12 salts, in which the Ca2* ily the case. When bladders were cultured in MEM-EBS in concentration had been increased to 1.8 mM (Fig. 2B). which all medium components were in higher concentra In addition to suppressing endophytic growth, elevated tion (1.4 times) than normal, excess hyperplasia occurred. Ca24 levels also produced a more organized epithelium. Epithelia became approximately 10 cell layers thick by 7 Even when compared with nonendophytic areas of epithelia days of culture. No endophytic growth developed, however. cultured in 0.3 mM Ca2' (Fig. 3A), epithelia cultured in 1.8 In another series of experiments, bladders were cultured mM Ca2' (Fig. 3B) had a more ordered cellular arrangement for 14 days, one half in F12 medium and the other half in and frequently displayed a more transitional morphology. F12M medium (8), which contains elevated levels of several Another characteristic of epithelia cultured in high Ca2' components including Ca24 (1.1 mM). In F12 medium, concentration was their reduced affinity for the underlying growth was endophytic (Fig. 5/4). In F12M medium no stroma. Epithelia cultured in 1.8 mM Ca2* had a tendency, endophytic growth occurred (Fig. 5B) and, although it was after 14 days of culture, to occasionally separate from the not possible quantitatively to compare the actual amount stroma (Fig. 4/4). These epithelia displayed a surprising of growth with that which occurred in F12 medium because affinity for the stainless steel grid, which resulted in occa of the extensive endophytic growth in F12, the extent of sional tearing of epithelia during the fixation process. growth in the 2 media appeared to be comparable. Epithelia cultured in 0.3 mM Ca24, on the other hand, Low Ca24 Concentration Stimulates Epithelial Cell Infil exhibited a high affinity for the underlying stroma. The tration into the Stroma. In view of the organizing effect epithelial-stromal borders of these epithelia (Fig. 4B) were that Ca24 exerted on the bladder epithelium in culture, the

MARCH 1978 587

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. D. H. Reese and R. D. Friedman so and qualitative aspects of growth of the bladder epithelium in organ culture. 80 The significant suppression of hyperplasia by Ca2' that we observed is quite interesting, since there have been 70 reports (1, 10, 18, 20) demonstrating that Ca2' stimulates cell proliferation at concentrations that we have found to i 60 be inhibitory. A discussion of why this discrepancy should Â§ 60 exist would be premature at this point, since there are numerous differences between our experiments and those showing stimulation by Ca2'. Three major points should be S emphasized about this study, however. ( a) The effect of S Ca2* concentration was measured on organ-cultured tissue in serum-free medium. ( b) In stating that Ca2' suppressed hyperplasia, we are not necessarily indicating that the rate of cell proliferation was reduced by Ca2'. An epithelium can become hyperplastic in essentially 2 ways, through an increased rate of proliferation or a decreased rate of matu 0.3 0.6 0.9 1.2 1.5 1.8 ration and cell sloughing from the surface. It is entirely CALCIUM CONCENTRATION (mM) possible, for example, that the rate of proliferation in high Chart 1. Effect of Ã‡a2'on hyperplasia. Explants were cultured for 12 Ca2* concentration was equal to or even greater than that days in MEM with F12 salts. Each point represents the mean number of in low Ca2' concentration, but that the rate of maturation nuclei per unit length (150 ^m) of epithelia from 4 or 5 expiants Â±S.E. Hatched area, mean values Â±S.E. for normal (uncultured) epithelia. was greater in high Ca2*. If, in the presence of high Ca2* effect of Ca2* concentrations lower than 0.3 mM was in concentration, the suppression of hyperplasia was due to the inhibition of cell proliferation, then our results can best vestigated to determine what the cellular behavior would be at abnormally low Ca2' concentrations. For these exper be interpreted in terms of the energy requirements for cell proliferation (see Ref. 3). Magnesium is a cofactor for the 3 iments, MEM-EBS containing 11 mM glucose and 2 mM transphosphorylation reactions of glycolysis. Ca2*, because sodium pyruvate was used. At Ca2' concentrations of 0.2 and 0.1 mM, growth was it competes with magnesium for either the required enzyme or the adenine nucleotide of these reactions, is capable of endophytic and there was no noticeable difference between inhibiting the reactions. Thus, it is possible that in the the growth characteristics of epithelia cultured at these presence of high Ca2' concentration, the bladder epithe concentrations and epithelia cultured at 0.3 mM Ca2*. At lium lacked sufficient energy to proliferate at the same rate 0.075 mw, on the other hand, there was a loss of cohesive- as that in low Ca2' concentration, (c) In this study the ness between cells in some regions of epithelia. This effect of Ca2* was measured on epithelial cells. Previous response was especially prominent in the basal region (Fig. studies showing Ca2' to be stimulatory to growth were on 6). Epithelial cells were no longer confined to the epithelium bone marrow- and thymus-derived cells (20) and fibroblasts but were observed either individually or as clusters in the (1, 10, 18). It is interesting, in connection with those lamina propria. Cellular degeneration and lytic activity was observations on fibroblasts, that in this study, although evident in the lamina propria and was confined only to that Ca2* had an inhibitory effect on the epithelium, stromal region of the stroma. There also appeared to be a decrease cells (presumably fibroblasts) appeared to be stimulated by in the number of connective tissue cells in the lamina Ca2* as evidenced by their accumulation at the epithelial- propria. With decreasing Ca2* concentrations, the extent stromal border in high Ca2' concentration and their appar of dissociation increased. At 0.025 mM Ca2*, a loss of ent reduction in numbers in the lamina propria in abnor cohesiveness between cells was evident over the entire mally low Ca2' concentration. thickness of the epithelium (Fig. 7). In epithelia cultured in The development of endophytic growth and its suppres Ca2'-free medium, there was extensive loss of cells due to sion by high Ca2* is most probably related to the involve sloughing; however, cells continued to be observed in the ment of Ca2* in maintaining cellular cohesiveness. Most basal region of the epithelium and in the stroma. cellular Ca2* is located on the cell surface or in the intercel Cell proliferation continued in epithelia cultured in low lular spaces, and has been shown to be very loosely bound Ca21. Occasional mitotic figures were observed in the basal (11). In the presence of 0.3 Ca2' in the medium, surface- region of the epithelium and in epithelial cells embedded bound Ca2*, which is at a concentration of about 1 mM (15), in the lamina propria. would be expected to be lost by simple gradient diffusion Cytologically, the most prominent feature of epithelial (11). This loss of Ca2* would result in lowered cohesive cells cultured in low Ca2' was the abnormal appearance of forces between epithelial cells. It is possible that the loss the nucleoli. Many were larger and more prominent than of cohesiveness creates a distortion in the normal direction normal, were frequently elongated, and appeared to be of the maturation process of the epithelium, such that cells associated with the nuclear membrane (Fig. 8). are allowed to grow down into the stroma. Growth per se does not appear to be a causative factor in the development DISCUSSION of endophytic growth, since conditions were found in which The results obtained in this study show the Ca2* concen excess growth occurred without endophytic growth. The tration to be a major determinant of both the quantitative stroma may also play an important role in blocking endo-

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phytic growth, since, as discussed above, fibroblasts ap REFERENCES peared to be stimulated by high Ca2* concentration. In the

presence of high Ca2* concentration, stromal elements may 1. Balk, D. S., Whitfield, J. F., Youdale, T., and Braun, A. C. Roles of Calcium, Serum, Plasma, and Folie Acid in the Control of Proliferation thus provide resistance to the downward growth of the of Normal and Rous Sarcoma Virus-infected Chicken Fibroblasts. Proc. epithelium. Nati. Acad. Sei. U. S., 70: 675-679, 1973. When the Ca2' concentration was decreased to 0.075 2. Bertram, J. S., and Craig, A. W. Specific Induction of Bladder Cancer in mM and below, there was considerable loss of cohesiveness Mice by Butyl-(4-hydroxybutyl)-nitrosamine and the Effects of Hormonal Modifications on the Sex Difference in Response. European J. Cancer, between epithelial cells, which resulted in cells dissociating 8: 587-594, 1972. from the main epithelial mass and infiltrating into the 3. Bygrave, F. L. The Ionic Environment and Metabolic Control. Nature, 214: 667-671, 1967. lamina propria. The presence of epithelial cells in the 4. Carruthers, C., and Suntzeff, V. The Role of Calcium in Carcinogenesis. lamina propria indicates a breakdown in the basement Science, 99: 245-247, 1944. 5. Chaves, E. Induction of Bladder Hyperplasia in Rats after a Single Dose membrane. This observation combined with the evidence of Cyclophosphamide. Rev. Franc. Etudes Clin. Biol., 13: 56-61, 1968. of lytic activity in the lamina propria suggests that epithelial 6. Coman, D. R. Decreased Mutual Adhesiveness, a Property of Cells from cells and/or fibroblasts may have elaborated proteolytic Squamous Cell Carcinomas. Cancer Res., 4: 625-629, 1944. 7. Coman, D. R. Adhesiveness and Stickiness: Two Independent Properties enzymes. These enzymes could come from degenerating of the Cell Surface. Cancer Res., 21: 1436-1438, 1961. cells or living cells, since it has been shown that the 8. Coon, H. G., and Weiss, M. C. A Quantitative Comparison of Formation removal of tissue Ca2' stimulates the leakage of proteins of Spontaneous and Virus-produced Viable Hybrids. Proc. Nati. Acad. Sei. U. S.,62: 852-859, 1969. from otherwise intact cells by increasing cell permeability 9. DeLong, R. P., Coman, D. R., and Zeidman. I. The Significance of Low (12). Calcium and High Potassium Content in Neoplastic Tissue. Cancer, 3: Although this study deals only with the effect of Ca2* in 718-721, 1950. vitro, it is likely that Ca2' is also a major factor in the 10. Dulbecco, R., and Elkington, J. Induction of Growth in Resting Fibro- blastic Cell Cultures by Ca". Proc. Nati. Acad. Sei. U. S., 72: 1584- control of growth in the bladder epithelium in vivo. This 1588,1975. 11. Hickie, R. A., and Kalant, H. Effects of Perfusion Media on the Ca' ' and contention is supported by in vivo observations which show Mg'* Content of Rat Liver. Can. J. Physiol. Pharmacol., 44: 893-900, that, under conditions of vitamin A deficiency, the bladder 1966. epithelium undergoes extensive hyperplasia with endophy- 12. Kalant. H., Murray, R. K., and Mons, W. Effect of EDTA on Leakage of tic growth (21) and the urinary excretion of Ca2t is drasti Proteins from Slices of Normal Rat Liver and DAB-induced Hepatoma. Cancer Res., 24: 570-581, 1964. cally reduced (22). 13. Koss, L. G. Tumors of the Urinary Bladder. Atlas of Tumor Pathology, There have been numerous reports in the literature show Fascicle 11, Ser. 2, pp. 5 and 103. Washington, D. C.: Armed Forces Institute of Pathology, 1975. ing that malignant cells have reduced affinity for each 14. Kunze, E., Schauer, A., and Schaft, S. Stages of Transformation in the other and contain lower amounts of Ca2" than normal cells Development of rV-Butyl-A/-(4-hydroxybutyl)-Nitrosamine-induced Tran (4, 6, 7, 9). In light of these earlier studies, our present sitional Cell Carcinomas in the Urinary Bladder of Rats. Z. Krebsforsch., 87 139-160, 1976. study, which shows a close similarity between the pattern 15. Rasmussen, H. Cell Communication, Calcium Ion, and Cyclic Adenosine of epithelial growth induced in vitro by low Ca2' concentra Monophosphate. Science, 770: 404-412, 1970. tion and the growth pattern that develops in vivo during 16. Reese, D. H., Friedman, R. D., Smith, J. M., and Sporn, M. B. Organ Culture of Normal and Carcinogen-treated Rat Bladder. Cancer Res., the preneoplastic phase of experimental bladder cancer, 36: 2525-2527, 1976. strongly suggests that there may be an early change in 17. Reese, D. H., Friedman, R. D., and Sporn, M. B. Induction of Hyperplasia some aspect of cellular Ca2' (probably decreased binding) and Its Suppression by Hydrocortisone in Organ-cultured Rat Urinary Bladder. Cancer Res., 37: 1421-1427, 1977. in epithelia and/or stroma that have been exposed to 18. Rubin, H., and Koide, T. Mutual Potentiation by Magnesium and Calcium bladder carcinogens. Furthermore, the ability to stimulate of Growth in Animal Cells. Proc. Nati. Acad. Sei. U. S., 73: 168-172. infiltrative growth by abnormally low Ca2' concentration in 1976. 19. Tiltman, A. J., and Frieden, G. H. The Histogenesis of Experimental the medium also suggests the possibility that invading Bladder Cancer. Invest. Urol., 9: 218-226, 1971. bladder cancer cells may have defects in control mecha 20. Whitfield, J. F., Rixon, R. H., MacManus, J. P., and Balk, S. D. Calcium, nisms which are normally regulated by Ca2*. Cyclic Adenosine 3'5'-Monophosphate, and the Control of Cell Prolifer ation: A Review. In Vitro, 8: 257-278, 1973. 21. Wolbach, S. B., and Howe, P. R. Tissue Changes Following Deprivation ACKNOWLEDGMENTS of Fat Soluble A Vitamin. J. Exptl. Med., 42: 753-777, 1925. We thank Margaret 0 Boyle for expert secretarial assistance and Dr. 22. Zile, M., DeLuca, H. F., and Ahrens, H. Vitamin A Deficiency and Hayden Coon for F12M medium. Urinary Calcium Excretion in Rats. J. Nutr., Ã•02: 1255-1258, 1972.

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All tissues were fixed in neutral formalin, embedded in glycol methacrylate. sectioned at 2 ^m, and stained with toluidine blue. Fig. 1. A, a half-bladder explant cultured for 14 days in MEM-EBS; B, other half of same bladder cultured 14 days in MEM with Ham's F12 salts, x 20. Fig. 2. Half-bladder expiants, from same bladder, cultured for 14 days in MEM with F12 salts. A. 0.3 mM Ã‡a2';B, 1.8mw Ã‡a2',x 20. Fig. 3. Higher power view of Fig. 2 showing differ ence in cellular organization. A, 0.3 mM Ca2'; note dysplasia in flat region of epithelium at left. Arrows, cells lining epithelial-stromal border. B, 1.8 mM Ca2*; note ordered transitional morphology, x 220.

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Ã¯Ã§Ã¯^rnÂ»Â»*'â€¢â€¢;-â€¢>Ã„.Â«I*>Â«Â¿â€¢ Â¿Ã€Ã®Ã¯fcfc&Vk'Ji Â«M5ÃŸ.Vi \&Â«iÃ¼Â¿Â¿$^^^.&5Ã¤i

**%^r -AÂ» * ' ?^r "^^ '*x^^^- *vj^"-' ^â€¢** ^CT w ^- '^."v A'*!â€¢//â€¢ ^*t $'*&'; !f ^: â€¢ -* '?*tV â€¢Â«f Fig. 4. Half-bladder expiants, from same bladder, ?s/*A-a^ Avwîo/'Y"A: it//* cultured for 14 days in MEM with F12 salts. A, 1.8 rnM Ca2*; note separation of epithelium from stroma and flattened cells (indicated by arrows) that were lining fferîÃÃo the epithelial-stromal border. B, 0.3 mw Ca2'; note is?-jf?|!*Ã„iVSP**^Ã¯^5!?îiPL^ absence of clearly defined epithelial-stromal border, â€¢'J5Wft5SÃÃ¡v:"â€¢1Â»t,v:^Â¿f x 330. Fig. 5. A, a half-bladder expiant cultured for 14 days in Ham's F12 medium; B, other half of same Ã' .iÃSfce '* Ã^'''â€¢Â»&w bladder cultured for 14 days in Ham's F12M medium; note extensive hyperplasia but absence of endophytic growth, x 20. n&Ã^$*$ . * ^v*-^r^ - Â»1 '"' ' -*'â€¢'-- - :'- 4B

MARCH 1978 591

Fig. 6. Expiant cultured for 14 days in 0.075 mw Ca2'. Medium was MEM-EBS containing 11 mw glu cose and 2 HIM sodium pyruvate. x 330. Fig. 7. Expiant cultured for 14 days in 0.025 mw Ca2". Details same as for Fig. 6. x 330. Fig. 8. Expiant cultured for 14 days in 0.075 mM Ca2'. Details same as for Fig. 6. x 540.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Suppression of Dysplasia and Hyperplasia by Calcium in Organ-cultured Urinary Bladder Epithelium

David H. Reese and Rosalind D. Friedman

Cancer Res 1978;38:586-592.

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